Ca++-ionophore-induced dissipation of intracellular proton gradients in rat thymocytes

Abstract

Rat thymocytes incorporate large amounts of acridine orange at concentrations of approximately 10 μM in about 10 minutes at 37^oC. The addition of (NH₄)₂SO₄ (at a final concentration of several mMolars) releases about 30% of the incorporated dye into the medium. The NH₄⁺-releasable dye uptake is almost completely abolished by 20 minutes' incubation with 4 μM of the Ca⁺⁺-ionophore A23187. Dye uptake is associated with an absorbance change at 492 mm and thus may be followed spectrophotometrically. However, NH₄⁺ at the above concentrations or nigericin (0.5 mg/ml) completely annul this change in absorbance, indicating that it reflects only the accumulation of the dye within the acidic cellular compartments. In a Ca⁺⁺-containing physiological saline, the addition of A23187 (at a final concentration of 8 μM) at the end of the dye uptake phase initiated a reversal in the absorbance change; in the absence of Ca⁺⁺, reversal occurred at a much lower rate. Incubation of cells for 30 minutes with 2 μM A23187 in Ca⁺⁺-containing saline completely abolished NH₄⁺-sensitive dye uptake; less A23187 (0.5 μM) and like or a longer incubation period brought about a striking decrease in NH₄⁺-sensitive dye uptake. Similar results were obtained with cells suspended in RPLM 1640 medium. In the absence of external Ca⁺⁺, A23187 impaired cell capacity to incorporate dye in a ΔpH-dependent manner, but a longer incubation time or higher concentrations of the ionophore were required to obtain a comparable effect. It is thus concluded that the ionophore dissipates intracellular pH gradients by an intracellular divalent cation (Ca⁺⁺ or also Mg⁺⁺)-H⁺ exchange.